The beauty of symmetry: siRNA phosphorodithioate modifications reduce stereocomplexity, ease analysis, and can improve in vivo potency

Abstract

Phosphorothioates (PSs) can be essential in stabilizing therapeutic oligonucleotides against enzymatic degradation. However, unless synthesis is performed with stereodefined amidites, each PS introduces a chemically undefined stereocenter, resulting in 2n unique molecules in the final product and affecting downstream analytics and purification. Replacing the second non-bridging oxygen with sulfur results in phosphorodithioate (PS2) linkages, thereby removing the stereocenter. We describe synthesis and analytical data for N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) with PS2 in the GalNAc cluster and at the siRNA termini. All siRNA conjugates with PS2 internucleotide linkages were produced with good yield and showed improved analytical properties. PS2 in the GalNAc cluster had no, or only minor, effect on in vitro and in vivo activity. Except for the 5′-antisense position, PS2 modifications were well tolerated at the siRNA termini, and a single PS2 internucleotide linkage gave similar or improved stabilization and in vitro activity as the two PSs typically used for end stabilization. Surprisingly, several of the PS2-containing siRNA conjugates resulted in increased in vivo activity and duration of action compared to the same siRNA sequence stabilized with PS linkages, suggesting PS2 linkages as interesting options for siRNA strand design with a reduced number of undefined stereocenters.